Human scFv antibody fragments specific for the epithelial tumour marker MUC-1, selected by phage display on living cells

New anti-cancer agents are being developed that specifically recognise tumour cells. Recognition is dependent upon the enhanced expression of antigenic determinants on the surface of tumour cells. The tumour exposure and the extracellular accessibility of the mucin MUC-1 make this marker a suitable target for tumour diagnosis and therapy. We isolated and characterised six human scFv antibody fragments that bound to the MUC-1 core protein, by selecting a large naive human phage display library directly on a MUC-1-expressing breast carcinoma cell line. Their binding characteristics have been studied by ELISA, FACS and indirect immunofluorescence. The human scFv antibody fragments were specific for the tandem repeat region of MUC-1 and their binding is inhibited by soluble antigen. Four human scFv antibody fragments (M2, M3, M8, M12) recognised the hydrophilic PDTRP region of the MUC-1 core protein, which is thought to be an immunodominant region. The human scFv antibody fragments were stable in human serum at 37 °C and retained their binding specificity. For imaging or targeting to tumours over-expressing MUC-1, it might be feasible to use these human scFv, or multivalent derivatives, as vehicles to deliver anti-cancer agents. Received: 2 November 2000 / Accepted: 11 January 2001     

An altered camelid-like single domain anti-idiotypic antibody fragment of HM-1 killer toxin: acts as an effective antifungal agent

Phage-display and competitive panning elution leads to the identification of minimum-sized antigen binders together with conventional antibodies from a mouse cDNA library constructed from HM-1 killer toxin neutralizing monoclonal antibody (nmAb-KT). Antigen-specific altered camelid-like single-domain heavy chain antibody (scFv K2) and a conventional antibody (scFv K1) have been isolated against the idiotypic antigen nmAb-KT. The objectives of the study were to examine (1) their properties as compared to conventional antibodies and also (2) their antifungal activity against different pathogenic and non-pathogenic fungal species. The alternative small antigen-binder, i.e., the single-domain heavy chain antibody, was originated from a conventional mouse scFv phage library through somatic hyper-mutation while selection against antigen. This single-domain antibody fragment was well expressed in bacteria and specifically bound with the idiotypic antigen nmAb-KT and had a high stability and solubility. Experimental data showed that the binding affinity for this single-domain antibody was 272-fold higher (K _d = 1.07 × 10⁻¹⁰ M) and antifungal activity was three- to fivefold more efficient (IC₅₀ = 0.46 × 10⁻⁶ to 1.17 × 10⁻⁶ M) than that for the conventional antibody (K _d = 2.91 × 10⁻⁸ M and IC₅₀ = 2.14 × 10⁻⁶ to 3.78 × 10⁻⁶ M). The derived single-domain antibody might be an ideal scaffold for anti-idiotypic antibody therapy and the development of smaller peptides or peptide mimetic drugs due to their less complex antigen-binding site. We expect that such single-domain synthetic antibodies will find their way into a number of biotechnological or medical applications.     

Pharmacokinetics and biodistribution of a light-chain-shuffled CC49 single-chain Fv antibody construct

Murine monoclonal antibodies to tumor-associated glycoprotein 72 (anti-TAG-72 mAb B72.3 and CC49) are among the most extensively studied mAb for immunotherapy of adenocarcinomas. They have been used clinically to localize primary and metastatic tumor sites; however, murine mAb generally induce potent human anti-(mouse antibody) responses. The immunogenicity of murine mAb can be minimized by genetic humanization of these antibodies, where non-human regions are replaced by the corresponding human sequences or complementary determining regions are grafted into the human framework regions. We have developed a humanized CC49 single-chain antibody construct (hu/muCC49 scFv) by replacing the murine CC49 variable light chain with the human subgroup IV germline variable light chain (Hum4 V_L). The major advantages of scFv molecules are their excellent penetration into the tumor tissue, rapid clearance rate, and much lower exposure to normal organs, especially bone marrow, than occur with intact antibody. The biochemical properties of hu/muCC49 scFv were compared to those of the murine CC49 scFv (muCC49 scFv). The association constants (K _a) for hu/muCC49 and muCC49 constructs were 1.1 × 10⁶ M⁻¹ and 1.4 × 10⁶ M⁻¹ respectively. Pharmacokinetic studies in mice showed similar rapid blood and whole-body clearance with a half-life of 6 min for both scFv. The biodistribution studies demonstrated equivalent tumor targeting to human colon carcinoma xenografts for muCC49 and hu/muCC49 scFv. These results indicate that the human variable light-chain subgroup IV can be used for the development of humanized or human immunoglobulin molecules potentially useful in both diagnostic and therapeutic applications with TAG-72-positive tumors. Received: 29 December 1999 / Accepted: 4 February 2000     

Construction, expression and characterisation of a single-chain diabody derived from a humanised anti-Lewis Y cancer targeting antibody using a heat-inducible bacterial secretion vector

 A single-chain antibody fragment (scFv) of the humanised monoclonal antibody, hu3S193, that reacts specifically with Le^y antigen expressed in numerous human epithelial carcinomas was constructed. A five-residue linker joined the C-terminus of the V_H and the N-terminus of the V_L, which prevented V-domain association into a monomeric scFv and instead directed non-covalent association of two scFvs into a dimer or diabody. The diabody was secreted into the E. coli periplasm using a heat-inducible vector, pPOW3, and recovered as a soluble, correctly processed protein, following osmotic shock or solubilised with 4M urea from the insoluble fraction. The diabody from both fractions was isolated by a rapid batch affinity chromatography procedure, using the FLAG affinity tag to minimise degradation and aggregation. The purified diabody has an M_r of ˜54 kDa, was stable and demonstrated similar binding activity as the parent monoclonal antibody, as measured by FACS and BIAcore analyses. The radiolabelled diabody showed a rapid tumour uptake, with fast blood clearance, proving it to be an excellent potential candidate as a tumour-imaging agent. Received: 16 November 2000 / Accepted: 8 March 2001     

Production and validation of the pharmacokinetics of a single-chain Fv fragment of the MGR6 antibody for targeting of tumors expressing HER-2

The HER-2 antigen, which is overexpressed in many breast carcinomas, is an ideal target for monoclonal antibodies due to its low expression in normal tissue and its homogeneous distribution in the tumor mass. We have developed and characterized the murine MAb MGR6 against HER-2, which is able to inhibit proliferation of tumor cells overexpressing HER-2. On the basis of these preclinical results, phase I studies in breast carcinoma patients were conducted and radiolocalization data indicated an antibody half life which directly paralleled that of other whole antibodies and thus resulting in a limited in vivo diagnostic capacity. To obtain a smaller reagent with possibly improved in vivo properties, a single chain variable fragment (scFv) of the original MGR6-producing hybridoma was generated by phage display technology. Biologically active MGR6 scFv was purified rapidly and at high yield by metal affinity chromatography. Competition FACS and ELISA analyses identified an epitope on the HER-2 extracellular domain that was shared by the scFv and the parental MAb. BIAcore analysis indicated a K_off of 9.3 × 10⁻⁴ s⁻¹, similar to that of the intact MGR6 MAb. Distribution and elimination half-lives of MGR6 scFv, calculated from in vivo preclinical evaluations, were much faster (13 min and 6.2 h, respectively) than previously published results for the intact MAb (mean t1/2β of 46 h). This represents a theoretical improvement in pharmacokinetics with respect to the parental murine MAb and points to the potential for utilizing this fragment in redirecting therapeutic agents, such as radioisotopes, to different human carcinomas overexpressing HER-2. Received: 10 August 2000 / Accepted: 19 October 2000     

Relationship between tumour morphology, antigen and antibody distribution measured by fusion of digital phosphor and photographic images

Antibody-directed cancer therapy has achieved encouraging responses despite poor localisation in tumour. This discrepancy may be attributed to heterogeneity of antibody delivery within tumours: preferential localisation in the better perfused and more radio- and chemosensitive areas provides a therapeutic advantage. Antibody distribution depends upon the interactions of many complex mechanisms. We have started to investigate this by studying the single and combined influence of two tumour-associated parameters, morphology and antigen, on antibody distribution. Tumours were taken from mice at 24 and 48 h after ¹²⁵I-labeled anti-CEA antibody injection. Images of antibody distribution, antigen distribution and tumour morphology were acquired by radioluminography, radioimmunoluminography and digitisation of morphology, respectively. Image registration allowed correlation of pixel values of antibody distribution with corresponding values of antigen distribution and morphology. At 24 h there was little correlation between antibody and antigen distribution, but strong positive correlation between antibody distribution and morphology, with preferential localisation in viable tumour areas. Correlation between antibody distribution and morphology fell significantly between 24 and 48 h, while that between antibody and antigen distribution remained low. However, the combination of morphology and antigen distribution showed the largest influence on antibody distribution. This novel technique demonstrates potential for combining multi-factor information in order to provide a greater understanding of antibody distribution in tumours, facilitating the optimisation of clinical treatments. Received: 29 November 2000 / Accepted: 11 January 2001     

Peptide derived from anti-idiotypic single-chain antibody is a potent antifungal agent compared to its parent fungicide HM-1 killer toxin peptide

Based on anti-idiotypic network theory in light of the need for new antifungal drugs, we attempted to identify biologically active fragments from HM-1 yeast killer toxin and its anti-idiotypic antibody and to compare their potency as an antifungal agent. Thirteen overlapping peptides from HM-1 killer toxin and six peptides from its anti-idiotypic single-chain variable fragment (scFv) antibodies representing the complementarity determining regions were synthesized. The binding affinities of these peptides were investigated and measured by Dot blot and surface plasmon resonance analysis and finally their antifungal activities were investigated by inhibition of growth, colony forming unit assay. Peptide P6, containing the potential active site of HM-1 was highly capable of inhibiting the growth of Saccharomyces cerevisiae but was less effective on pathogenic fungi. However, peptide fragments derived from scFv antibody exerted remarkable inhibitory effect on the growth of pathogenic strains of Candida and Cryptococcus species in vitro. One scFv-derived decapeptide (SP6) was selected as the strongest killer peptide for its high binding affinity and antifungal abilities on both Candida and Cryptococcus species with IC₅₀ values from 2.33 × 10⁻⁷ M to 36.0 × 10⁻⁷ M. SP6 peptide activity was neutralized by laminarin, a β-1,3-glucan molecule, indicating this peptide derived from scFv anti-idiotypic antibody retains antifungal activity through interaction with cell wall β-glucan of their target fungal cells. Experimental evidence strongly suggested the possibility of development of anti-idiotypic scFv peptide-based antifungal agents which may lead to improve therapeutics for the management of varieties of fungal infections.     

In vitro characterization of a recombinant 32P-phosphorylated anti-(carcinoembryonic antigen) single-chain antibody

 The major limitations of monoclonal antibody conjugates as therapeutic agents have been their poor tumour targeting, inadequate tumour penetration and immunogenicity. More even and deeper tissue penetration has been demonstrated with smaller antibody fragments. The smaller size and absence of an Fc segment may contribute to a lowered immunogenicity with single-chain antibodies (scFv) and also permit their recombinant engineering and bacterial expression. We describe the successful engineering, expression and pre-clinical characterisation of a phosphorylatable “kemptide” (Leu-Arg-Arg-Ala-Ser-Gly) anti-carcinoembryonic antigen (anti-CEA) scFv (PKS-scFv), for use as a radioimmunotherapeutic agent. Specifically, a yield of 6 mg/l induced culture was obtained. Site-specific phosphorylation was demonstrated without loss of specificity. In vitro assays revealed a selective cytotoxicity of ³²P-PKS-scFv for high-CEA-expressing LS-174T cells compared to the low-CEA-expressing HT-29 cells, with a rapid internalisation rate. Received: 20 March 1997 / Accepted: 5 February 1998     

Generation of a stable anti-human CD44v6 scFv and analysis of its cancer-targeting ability in vitro

CD44v6 is a cancer-associated antigen that mainly expresses in a subset of adenocarcinomas. Therefore, in this study, anti-human CD44v6 single-chain variable fragment (scFv) has been selected and characterized because it is the first step of primary importance towards the construction of a novel cancer-targeted agent for cancer diagnosis and therapy. In our study, anti-human CD44v6 scFv was selected from a human phage-displayed scFv library based on its ability to bind in vitro to CD44v6 antigen. Subsequently, immunofluorescent staining and Western blot analyses were performed to measure the binding characteristics of this scFv. In addition, flow cytometric analysis was done to verify its cancer-targeting ability in vitro. And a flow cytometry-based assay was used to determine its equilibrium dissociation constant (K _D). Finally, one functional anti-CD44v6 scFv was selected and characterized. Nucleotide sequencing verified that it was an incomplete scFv gene but had a variable heavy chain (V_H) alone. However, anti-CD44v6 scFv demonstrated cell-binding and antigen-binding activities by immunofluorescent staining and Western blot analyses. Furthermore, flow cytometric analysis proved that this scFv specifically targeted CD44v6-expressing cancer cells other than CD44v6 non-expressing normal cells or tumor cells in vitro. The K _D of this scFv was calculated to be 7.85 ± 0.93 × 10⁻⁸ M. In summary, the selected human scFv against CD44v6 has specific binding activity and favorable binding affinity despite lacking a variable light chain (V_L). Moreover, it can effectively and specifically target CD44v6-expressing cancer cells. All these characteristics make anti-CD44v6 scFv a promising agent for cancer detection and anti-cancer therapy.     

Serum heat shock protein 27 antigen and antibody levels appear to be related to the macrovascular complications associated with insulin resistance: a pilot study

Heat shock protein 27 (Hsp27) is over-expressed when cells are exposed to stressful conditions that include oxidative stress. Oxidative stress has been implicated in the pathogenesis of cardiovascular disease (CVD), diabetes and insulin resistance. We have investigated the concentrations of serum Hsp27 antigen and antibodies in subjects from different glycaemic categories, who either did or did not have established CVD. Serum Hsp27 antigen and antibody levels (immunoglobulins M and G (IgM and IgG)) were determined by enzyme-linked immunosorbent assays (ELISAs) in 68 individuals: 26 with normal glucose tolerance (NGT), 10 with (+) and 16 without (−) a history of CVD and 42 individuals with varying degrees of glucose intolerance (GI; 21 with and 21 without a history of CVD). Insulin sensitivity was determined in each subject using indices derived from the homeostasis model assessment of sensitivity and the insulin sensitivity index for glycaemia. Serum Hsp27 concentrations were significantly higher in GI (+CVD) subjects compared to GI (−CVD) subjects (p = 0.03), NGT (−CVD) subjects (p = 0.02) and NGT (+CVD) subjects (p = 0.04) and were positively correlated to fasting plasma glucose for all subjects (r = 0.28, p = 0.03). IgM antibody levels were significantly higher in GI (+CVD) subjects compared to NGT (−CVD) group (p = 0.02) and were inversely related to fasting insulin concentrations (r = −0.27, p = 0.04) and the 2-h insulin concentrations (r = −0.29, p = 0.03) for all subjects. Serum IgG antibody levels were higher in GI (+CVD) group compared to GI (−CVD) group (p = 0.06). In conclusion, Hsp27 and its antibody concentrations appear to relate to the presence of cardiovascular complications in patients with GI.     

Biodistribution of 111In-labelled engineered human antibody CTM01 (hCTM01) in ovarian cancer patients: influence of prior administration of unlabelled hCTM01

mAb hCTM01 binds a carcinoma-associated antigen, the MUC1 gene product. The antigen is also present in the circulation, and administration of ¹¹¹In-labelled hCTM01 results in the formation of immune complexes with enhanced accumulation in the liver. To avoid the unwanted effect of circulating radioactive immune complexes, a strategy to remove the circulating antigen was investigated using a split-dosage schedule. Eleven patients suspected of having ovarian carcinoma were injected with 1 mg/kg unlabelled hCTM01, 1 h before receiving 0.1 mg/kg ¹¹¹In-labelled hCTM01 (100 MBq). The amount of radioactivity was determined in resected tumour tissue, various normal tissues and blood samples obtained at laparotomy 6 days postinjection (p.i.). In all patients, the circulating antigen decreased to its nadir after the unlabelled antibody infusion and immune complex formation was demonstrated. Uptake in tumour deposits 6 days p.i. was 11.1 times higher than in normal tissues (P<0.0001) and 5.9 times higher than in blood (P<0.0001). ¹¹¹In activity in liver tissue was comparable to ¹¹¹In uptake in tumour tissue, and considerably lower than previously reported in patients not pretreated with unlabelled antibody. The split-dosing strategy would appear to be advantageous for use of hCTM01 as a specific carrier for the delivery of cytotoxic agents to patients with ovarian cancer. Received: 12 February 1998 / Accepted: 30 April 1998     

Control of Established Colon Cancer Xenografts Using a Novel Humanized Single Chain Antibody-Streptococcal Superantigen Fusion Protein Targeting the 5T4 Oncofetal Antigen

Superantigens (SAgs) are microbial toxins that cross-link T cell receptors with major histocompatibility class II (MHC-II) molecules leading to the activation of large numbers of T cells. Herein, we describe the development and preclinical testing of a novel tumor-targeted SAg (TTS) therapeutic built using the streptococcal pyrogenic exotoxin C (SpeC) SAg and targeting cancer cells expressing the 5T4 tumor-associated antigen (TAA). To inhibit potentially harmful widespread immune cell activation, a SpeC mutation within the high-affinity MHC-II binding interface was generated (SpeC_D203A) that demonstrated a pronounced reduction in mitogenic activity, yet this mutant could still induce immune cell-mediated cancer cell death in vitro. To target 5T4⁺ cancer cells, we engineered a humanized single chain variable fragment (scFv) antibody to recognize 5T4 (scFv5T4). Specific targeting of scFv5T4 was verified. SpeC_D203A fused to scFv5T4 maintained the ability to activate and induce immune cell-mediated cytotoxicity of colorectal cancer cells. Using a xenograft model of established human colon cancer, we demonstrated that the SpeC-based TTS was able to control the growth and spread of large tumors in vivo. This required both TAA targeting by scFv5T4 and functional SAg activity. These studies lay the foundation for the development of streptococcal SAgs as ‘next-generation’ TTSs for cancer immunotherapy.     

Phage-selected primate antibodies fused to superantigens for immunotherapy of malignant melanoma

 The high-molecular-weight melanoma-associated antigen, HMW-MAA, has been demonstrated to be of potential interest for diagnosis and treatment of malignant melanoma. Murine monoclonal antibodies (mAb) generated in response to different epitopes of this cell-surface molecule efficiently localise to metastatic lesions in patients with disseminated disease. In this work, phage-display-driven selection for melanoma-reactive antibodies generated HMW-MAA specificities capable of targeting bacterial superantigens (SAg) and cytotoxic T cells to melanoma cells. Cynomolgus monkeys were immunised with a crude suspension of metastatic melanoma. A strong serological response towards HMW-MAA demonstrated its role as an immunodominant molecule in the primate. Several clones producing monoclonal scFv antibody fragments that react with HMW-MAA were identified using melanoma cells and tissue sections for phage selection of a recombinant antibody phage library generated from lymph node mRNA. One of these scFv fragments, K305, was transferred and expressed as a Fab-SAg fusion protein and evaluated as the tumour-targeting moiety for superantigen-based immunotherapy. It binds with high affinity to a unique human-specific epitope on the HMW-MAA, and demonstrates more restricted crossreactivity with normal smooth-muscle cells than previously described murine mAb. The K305 Fab was fused to the superantigen staphylococcal enterotoxin A (D227A) [SEA(D227A)], which had been mutated to reduce its intrinsic MHC class II binding affinity, and the fusion protein was used to demonstrate redirection of T cell cytotoxicity to melanoma cells in vitro. In mice with severe combined immunodeficiency, carrying human melanoma tumours, engraftment of human lymphoid cells followed by treatment with the K305Fab-SEA(D227A) fusion protein, induced HMW-MAA-specific tumour growth reduction. The phage-selected K305 antibody demonstrated high-affinity binding and selectivity, supporting its use for tumour therapy in conjunction with T-cell-activating superantigens. Received: 9 September 1999 / Accepted: 21 October 1999     

The effect of T1 and T2 cytokines on the cytotoxic T cell response to mannan-MUC1

MUC1 is a mucin over-expressed in breast cancer and a proposed target for immunotherapy. By immunising mice with MUC1 conjugated to mannan (M-FP), CD8⁺ MHC-class-I restricted cytotoxic T lymphocytes (CTL), of high CTL precursor (CTLp) frequency (1/8000) and with significant tumour protection, can be induced. The effect of various cytokines [interleukin-2 (IL-2), IL-4, IL-6, IL-7, interferon γ (IFNγ), and granulocyte/macrophage-colony-stimulating factor (GM-CSF)] on the MUC1 CTL immune response was investigated (a) by measuring the frequencies of CTLp in mice immunised with vaccinia virus constructs containing recombinant cytokines and M-FP, or (b) by immunising cytokine- or cytokine-receptor-knockout (−/−) mice with M-FP. Vaccinia virus (VV) constructs containing recombinant cytokines were used either individually or in combination in vivo with M-FP immunisation. M-FP immunisations combined with VV-IL-2, VV-IL-7 and VV-GM-CSF, and combinations of VV-IFNγ + VV-IL-2, VV-IFNγ + VV-IL-4 or VV-GM-CSF + VV-IL-7 increased CTLp frequencies up to threefold (1/17 666: M-FP + VV-GM-CSF + VV-IL-7) compared to M-FP (1/77 500) alone. By contrast, M-FP combined with VV-IL-4 decreased the CTLp frequency threefold whereas VV-IL-6 and VV-IFNγ had no effect. Studies in cytokine- and cytokine-receptor-gene-knockout (−/−) mice demonstrated that mice that are IL-2 −/− and IL-7 receptor −/− produce the same CTLp response to M-FP as do control mice, whereas responses in the IL-6 −/−, IL-10 −/− and IFNγ−/− mice were marginally improved and responses to M-FP in IL-4 −/− and tumour necrosis factor receptor 2 −/− mice were weaker. In spite of the increase in CTLp frequency, this was not reflected in an in vivo tumour model. Tumour challenges using MUC1⁺ P815 cells, demonstrated that the addition of cytokines had little additive effect on the already effective tumour-regression capabilities of M-FP alone. Received: 24 September 1998 / Accepted: 21 September 1999     

4-Chlorophenol degradation by a bacterial consortium: development of a granular activated carbon biofilm reactor

A bacterial consortium that can degrade chloro- and nitrophenols has been isolated from the rhizosphere of Phragmitis communis. Degradation of 4-chlorophenol (4-CP) by a consortium attached to granular activated carbon (GAC) in a biofilm reactor was evaluated during both open and closed modes of operation. During the operation of the biofilm reactor, 4-CP was not detected in the column effluent, being either adsorbed to the GAC or biodegraded by the consortium. When 4-CP at 100 mg l⁻¹ was fed to the column in open mode operation (20 mg g⁻¹ GAC total supply), up to 27% was immediately available for biodegradation, the rest being adsorbed to the GAC. Biodegradation continued after the system was returned to closed mode operation, indicating that GAC bound 4-CP became available to the consortium. Biofilm batch cultures supplied with 10–216 mg 4-CP g⁻¹ GAC suggested that a residual fraction of GAC-bound 4-CP was biologically unavailable. The consortium was able to metabolise 4-CP after perturbations by the addition of chromium (Cr VI) at 1–5 mg l⁻¹ and nitrate at concentrations up to 400 mg l⁻¹. The development of the biofilm structure was analysed by scanning electron microscopy and confocal laser scanning microscopy (CLSM) techniques. CLSM revealed a heterogeneous structure with a network of channels throughout the biofilm, partially occupied by microbial exopolymer structures. Received: 17 March 1999 / Received revision: 27 May 1999 / Accepted: 28 May 1999     

Single-chain antibodies against human insulin-like growth factor I receptor: expression, purification, and effect on tumor growth

Insulin-like growth factors (IGF) I and II are potent mitogens for a variety of cancer cells. The proliferative and anti-apoptotic actions of IGF are mediated by the IGF-I receptor (IGF-IR), to which both IGF-I and IGF-II bind with high affinity. To investigate the mitogenic and anti-apoptotic activities of IGF-IR and to achieve better inhibition of IGF-IR function, single-chain antibodies against human IGF-IR (αIGF-IR scFvs) were constructed and expressed. IgG cDNA encoding variable regions of light and heavy chains (V_L and V_H) from mouse IgG were cloned from a hybridoma producing the 1H7 αIGF-IR monoclonal antibody [Li et al., Biochem Biophys Res Commun 196: 92–98 (1993)]. The splice-overlap extension polymerase chain reaction was used to assemble a gene encoding the αIGF-IR scFv, including the N-terminal signal peptide, V_L, linker peptide, V_H, and C-terminal DYKD tag. Two types of soluble αIGF-IR scFvs, a prototype αIGF-IR scFv and its alternative type αIGF-IR scFv-Fc, were constructed and expressed in murine myeloma cells. αIGF-IR scFv-Fc, containing the human IgG1 Fc domain, was stably expressed in NS0 myeloma cells, using a glutamine synthase selection system, and purified from the conditioned medium of stable clones by protein-A–agarose chromatography. Levels of αIGF-IR scFv-Fc expression ranged from 40 mg/l to 100 mg/l conditioned medium. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis under reducing and nonreducing conditions indicated that αIGF-IR scFv-Fc is a dimeric antibody. αIGF-IR scFv-Fc retained general characteristics of the parental 1H7 monoclonal antibody except that its binding affinity for IGF-IR was estimated to be approximately 10⁸ M⁻¹, which was one-order of magnitude lower than that of 1H7 monoclonal antibody. Injection of αIGF-IR scFv-Fc (500 μg/mouse, twice a week) significantly suppressed MCF-7 tumor growth in athymic mice. These results suggest that the αIGF-IR scFv-Fc is a first-generation recombinant αIGF-IR for the potential development of future αIGF-IR therapeutics. Received: 21 January 2000 / Accepted: 7 March 2000     

Biodegradation of propanol and isopropanol by a mixed microbial consortium

The aerobic biodegradation of high concentrations of 1-propanol and 2-propanol (IPA) by a mixed microbial consortium was investigated. Solvent concentrations were one order of magnitude greater than any previously reported in the literature. The consortium utilized these solvents as their sole carbon source to a maximum cell density of 2.4 × 10⁹ cells ml⁻¹. Enrichment experiments with propanol or IPA as carbon sources were carried out in batch culture and maximum specific growth rates (μ_max) calculated. At 20 °C, μ _max values were calculated to be 0.0305 h⁻¹ and 0.1093 h⁻¹ on 1% (v/v) IPA and 1-propanol, respectively. Growth on propanol and IPA was carried out between temperatures of 10 °C and 45 °C. Temperature shock responses by the microbial consortium at temperatures above 45 °C were demonstrated by considerable cell flocculation. An increase in propanol substrate concentration from 1% (v/v) to 2% (v/v) decreased the μ _max from 0.1093 h⁻¹ to 0.0715 h⁻¹. Maximum achievable biodegradation rates of propanol and IPA were 6.11 × 10⁻³% (v/v) h⁻¹ and 2.72 × 10⁻³% (v/v) h⁻¹, respectively. Generation of acetone during IPA biodegradation commenced at 264 h and reached a maximum concentration of 0.4% (v/v). The results demonstrate the potential of mixed microbial consortia in the bioremediation of solvent-containing waste streams. Received: 14 December 1999 / Received revision: 3 April 2000 / Accepted: 7 April 2000     

The Thomsen-Friedenreich disaccharide as antigen for in vivo tumor targeting with multivalent scFvs

The Thomsen-Friedenreich disaccharide (TF_α) is a promising antigen for tumor immunotargeting, since it is almost exclusively expressed on carcinoma tissues. So far, an obstacle preventing the exploitation of TF for immunotargeting has been the lack of suitable (non-IgM) antibodies with high affinity and specificity. Recently we reported on a novel strategy for generating antibodies toward small uncharged carbohydrates and the generation of recombinant antibodies toward TF. Among them, two multivalent scFv antibodies showed sub-micromolar functional affinities and appeared well suited for immunotargeting. In the present study, the trimeric scFv(1aa) and the tetrameric scFv(0aa) have been further developed for radioimmunotargeting. The scFvs were radiolabeled with ¹¹¹In using DTPA as chelator without losing binding activity or molecular stoichiometry. Binding affinities as high as 1 × 10⁻⁷ M toward TF displayed on living cells were determined. Antibody biodistribution and tumor targeting efficacy were studied in TF-positive human breast cancer (ZR-75-1) bearing mice. TF was successfully targeted in vivo with tumor uptakes of ∼11 and 8% ID/g after 24 h for the trimeric and tetrameric scFv, respectively. These results validate TF as a potent antigen for tumor targeting. The biodistribution of the scFvs was comparable to that reported for IgGs. In contrast to the IgGs, the serum clearance of the scFvs was very fast, which could be an advantage in a therapeutic setting. Furthermore, kidney uptake, which is often critical for small recombinant antibodies labeled with radio-metals, was low with the tetramer (11% ID/g). We conclude that the multimeric anti-TF scFvs are promising candidates to be further developed toward therapeutic application. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Toluene vapour removal in a laboratory-scale biofilter

A bench-scale biofilter with a 0.5-m high filter bed, inoculated with a toluene-degrading strain of Acinetobacter sp. NCIMB 9689, was used to study toluene removal from a synthetic waste air stream. Different sets of continuous tests were conducted at influent toluene concentrations ranging over 0.1–4.0 g m⁻³ and at superficial gas velocities ranging over 17.8–255 m h⁻¹. The maximum volumetric toluene removal rate for the biofilter (242 g m⁻³ h⁻¹) was obtained at a superficial gas velocity of 127.5 m h⁻¹ (corresponding to a residence time of 28 s) and a toluene inlet concentration of 4.0 g m⁻³. Under these operating conditions, toluene removal efficiency was only 0.238, which suggested that effective operation required higher residence times. Removal efficiencies higher than 0.9 were achieved at organic loads less than 113.7 g m⁻³ h⁻¹. A macro-kinetic study, performed using concentration profiles along the bioreactor, revealed this process was limited by diffusion at organic loads less than 100 g m⁻³ h⁻¹ and by biological reaction beyond this threshold. Received: 10 October 1999 / Received revision: 15 February 2000 / Accepted: 18 February 2000     

Effects of 3 days of carbohydrate supplementation on muscle glycogen content and utilisation during a 1-h cycling performance

This study compared the effects of supplementing the normal diets of six trained cyclists [maximal oxygen uptake O_2max) 4.5 (0.36)l · min⁻¹; values are mean (SD)] with additional carbohydrate (CHO) on muscle glycogen utilisation during a 1-h cycle time-trial (TT). Using a randomised crossover design, subjects consumed either their normal diet (NORM) for 3 days, which consisted of 426 (137) g · day⁻¹ CHO [5.9 (1.4) g · kg⁻¹ body mass (BM)], or additional CHO (SUPP) to increase their intake to 661 (76) g · day⁻¹ [9.3 (0.7) g · kg⁻¹ BM]. The SUPP diet elevated muscle glycogen content from 459 (83) to 565 (62) mmol · kg⁻¹ dry weight (d.w.) (P < 0.05). However, despite the increased pre-exercise muscle glycogen stores, there was no difference in the distance cycled during the TT [40.41 (1.44) vs 40.18 (1.76) km for NORM and SUPP, respectively]. With NORM, muscle glycogen declined from 459 (83) to 175 (64) mmol · kg⁻¹ d.w., whereas with SUPP the corresponding values were 565 (62) and 292 (113) mmol · kg⁻¹ d.w. Accordingly, both muscle glycogen utilisation [277 (64) vs 273 (114) mmol · kg⁻¹ d.w.] and total CHO oxidation [169 (20) vs 165 (30) g · h⁻¹ for NORM and SUPP, respectively] were similar. Neither were there any differences in plasma glucose or lactate concentrations during the two experimental trials. Plasma glucose concentration averaged 5.5 (0.5) and 5.6 (0.6) mmol · l⁻¹, while plasma lactate concentration averaged 4.4 (1.9) and 4.4 (2.3) mmol · l⁻¹ for NORM and SUPP, respectively. The results of this study show that when well-trained subjects increase the CHO content of their diet for 3 days from 6 to 9 g · kg⁻¹ BM there is only a modest increase in muscle glycogen content. Since supplementary CHO did not improve TT performance, we conclude that additional CHO provides no benefit to performance for athletes who compete in intense, continuous events lasting 1 h. Furthermore, the substantial muscle CHO reserves observed at the termination of exercise indicate that whole-muscle glycogen depletion does not determine fatigue at this exercise intensity and duration. Accepted: 25 November 1996